JP5389930B2 - Pseudoproline dipeptide - Google Patents

Description

  The present invention has the formula

で示される化合物の新規な製造方法に関するものである。

It is related with the novel manufacturing method of the compound shown by these.

  The pseudoproline dipeptide of formula I can be used as a reversible protecting group for Ser, Thr, and Cys and is a versatile means to overcome some inherent problems in the field of peptide chemistry. It has been found [JACS 1996, 118, 9218-9227]. The presence of ψPro in the peptide sequence results in the destruction of the β sheet structure, which is regarded as the cause of intermolecular aggregation. The resulting increased solvation and coupling kinetics in peptide assembly, such as Fmoc solid phase peptide synthesis, facilitate chain extension, especially for peptides with “difficult sequences”.

  A synthetic approach to pseudoproline dipeptide is disclosed in International Publication No. 2008/000641, which is a PCT publication. Obtaining the compound of formula I

［式中、Ｒ^１、Ｒ^２、Ｒ^５、Ｒ^６、Ｒ^７及びＲ^８は、上記ＰＣＴ公報に定義されている］で示されるアンモニウム塩中間体を経由して達成される。

[Wherein R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ and R ⁸ are defined as defined in the above PCT publication].

  One major disadvantage of the approaches known in the art is that the dipeptide needs to be purified by isolation of its ammonium salt intermediate, which must be released to the dipeptide prior to ring closure. This synthesis was therefore found to be unsuitable for industrial scale applications.

  The object of the present invention is to provide a concise and technically feasible synthesis of the pseudoproline dipeptide of formula I which makes it possible to obtain the product in high yield.

  The object was achieved by the method outlined below. formula

［式中、Ｒ^１は、α−アミノ酸の側鎖であり、Ｒ^２は、アミノ保護基であり、Ｒ^３及びＲ^４は、独立して水素（但し、Ｒ^３及びＲ^４の両方ともが水素ではない）又はＣ_１−４−アルキルより選択され、Ｒ^５は、水素又はメチルである］で示される化合物の製造方法であって、
ａ）式

[Wherein R ¹ is the side chain of the α-amino acid, R ² is an amino protecting group, R ³ and R ⁴ are independently hydrogen (provided that both R ³ and R ⁴ are Is not hydrogen) or C _1-4 -alkyl, and R ⁵ is hydrogen or methyl],
a) Formula

［式中、Ｒ^１及びＲ^２は上記と同義である］
で示されるアミノ酸誘導体を、セリン又はトレオニンと共に、式

[Wherein, R ¹ and R ² are as defined above]
Together with serine or threonine, the amino acid derivative represented by

で示されるジペプチドに変換すること（それに関して活性化剤として水溶性カルボジイミドを使用する）、及び
ｂ）酸触媒の存在下で、式

In which the water-soluble carbodiimide is used as an activator, and b) in the presence of an acid catalyst, the formula

［式中、Ｒ^３及びＲ^４は、独立して水素又はＣ_１−４−アルキルより選択されるが、但し、Ｒ^３及びＲ^４の両方ともが水素ではなく、Ｒ^９ａ及びＲ^９ｂは、独立してＣ_１−４−アルキルである］で示される化合物と共に式ＩＩＩで示されるジペプチドの閉環を実施すること
を含む。

[Wherein R ³ and R ⁴ are independently selected from hydrogen or C _1-4 -alkyl, provided that both R ³ and R ⁴ are not hydrogen and R ^9a and R ^9b are Carrying out the ring closure of the dipeptide of formula III together with a compound of the formula independently of C _1-4 -alkyl].

  Furthermore, it is understood that serine or threonine can be used in either its L- or D-form, as a racemate, or in various mixtures of its isomers. Preferably, L-type is used.

The term “C _1-4 -alkyl” represents a branched or straight-chain monovalent saturated aliphatic hydrocarbon group of 1 to 4 carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl and t-butyl.

The term “amino acid side chain” used for the substituent R ¹ is in particular valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamine, glutamic acid, histidine, lysine, arginine, aspartic acid, alanine, serine, threonine. Represents a side chain of an α-amino acid selected from tyrosine, tryptophan, cysteine, glycine, aminoisobutyric acid and proline.

  In the side chain of an amino acid bearing a hydroxy group, the hydroxy group is optionally protected by a hydroxy protecting group as defined below. In the side chain bearing an additional amino group, the amino group is optionally protected by an amino protecting group as defined below.

R ¹ preferably represents the side chains of valine, leucine, isoleucine, phenylalanine, asparagine, glutamine, glutamic acid, lysine, aspartic acid, alanine, serine, threonine, tyrosine and tryptophan. In a further preferred embodiment, R ¹ represents a serine or threonine side chain.

The term “amino protecting group” refers to any substituent conventionally used to prevent the reactivity of an amino group. Suitable amino protecting groups are described in Green T., “Protective Groups in Organic Synthesis”, Chapter 7, John Wiley and Sons, Inc., 1991, 309-385. A suitable amino protecting group defined under R ² should withstand acidic conditions. Preferably, Fmoc, Z, Moz, Troc, Teoc or Voc, more preferably Fmoc is used.

  The term “hydroxy protecting group” refers to any substituent conventionally used to prevent the reactivity of a hydroxy group. Suitable hydroxy protecting groups are described in Green T., “Protective Groups in Organic Synthesis”, Chapter 1, John Wiley and Sons, Inc., 1991, 10-142. Suitable hydroxy protecting groups are t-butyl, benzyl, TBDMS or TBDPS. A preferred hydroxy protecting group is t-butyl.

  The meanings of the abbreviations used in the specification and the claims are as outlined in the table below:

Step a)
In the first step a), the formula

［式中、Ｒ^１及びＲ^２は、上記と同義である］
で示されるアミノ酸誘導体をセリン又はトレオニンと共に式

[Wherein, R ¹ and R ² are as defined above]
Together with serine or threonine

で示されるジペプチドに変換する（それに関して活性化剤として水溶性カルボジイミドを使用する）。

(For which water-soluble carbodiimide is used as activator).

The amino acid derivative of formula II is usually a commercially available compound. Depending on the choice for R ¹ and R ² , a suitable amino acid derivative of formula II is Fmoc-L-Ser (tBu) —OH, or Fmoc-L-Thr (tBu) —OH.

  A suitable water soluble carbodiimide activator is EDC or EAC or a salt thereof, preferably the hydrochloride of EDC.

  Usually the water soluble carbodiimide activator is applied together with a further activator selected from HOSu or HOBt.

  A preferred activator is EDC.HCl / HOSu.

  For one equivalent of the amino acid derivative of formula II, EDC is usually used at 1.0 to 1.5 equivalents and HOSu is usually used at 1.0 to 1.5 equivalents.

  Usually, the activation reaction is carried out in the presence of a suitable organic solvent such as ethyl acetate, N, N-dimethylformamide, acetone or tetrahydrofuran, preferably tetrahydrofuran and / or N, N-dimethylformamide at -10 ° C to 25 ° C. At a temperature of

  Next, the coupling with serine or threonine, preferably L-serine or L-threonine can be performed at a temperature of -10 ° C to 25 ° C in the presence of an inorganic base.

  Usually the coupling is performed by adding the activated ester solution obtained from the activation reaction to an aqueous suspension of serine or threonine and an inorganic base.

  Suitable inorganic bases are alkali carbonates such as lithium, sodium or potassium carbonate or hydroxides or mixtures thereof.

  Preferred is lithium carbonate and / or lithium hydroxide, and more preferred is a mixture of lithium carbonate and lithium hydroxide.

  Inorganic bases are usually used in a stoichiometric ratio with respect to serine or threonine.

  The ratio of serine or threonine: amino acid derivative of formula II is usually selected in the range of 1.5 to 4.0: 1, preferably 2.0 to 3.0: 1.

  The pH of the reaction mixture is appropriately maintained in the range of 7.5 to 9.5.

  After the conversion is complete, the reaction mixture is acidified with mineral acid. Suitable mineral acids are aqueous sulfuric acid or aqueous HCl, preferably aqueous sulfuric acid.

  The dipeptide of formula III can be isolated according to methods known to those skilled in the art. In a preferred embodiment of the invention, the dipeptide of formula III is used directly in process step b) without isolation.

Step b)
Step b) has the formula

［式中、Ｒ^３、Ｒ^４、Ｒ^９ａ及びＲ^９ｂは、上記と同義である］
で示される化合物と共に式ＩＩＩで示されるジペプチドの閉環を酸触媒の存在下で、実施することを必要とする。

[Wherein R ³ , R ⁴ , R ^9a and R ^9b are as defined above]
It is necessary to carry out the ring closure of the dipeptide of the formula III together with the compound of the formula in the presence of an acid catalyst.

  Preferably, ring closure is performed with 2,2-dimethoxypropane. Ideally, the compound of formula IV is used in an amount of 6.0 to 16.0 equivalents, preferably 7.0 to 12.0 equivalents, relative to the dipeptide obtained in step b).

  In a preferred embodiment, 2,2-dimethoxypropane is continuously added to the reaction mixture, and the generated methanol is continuously distilled off in parallel.

  The reaction temperature is usually maintained in the range of 15 ° C to 35 ° C, preferably 20 ° C to 30 ° C.

  Suitable acid catalysts are selected from methanesulfonic acid, (+) camphor-10-sulfonic acid, p-toluenesulfonic acid or pyridinium p-toluenesulfonate, with methanesulfonic acid being preferred. The acid catalyst is usually used in an amount of 0.05 to 0.30 equivalents, preferably 0.08 to 0.15 equivalents, relative to the dipeptide of formula III obtained in step b).

  Ideally, the organic solvent applied for the conversion in step b) is substantially free of water. A suitable solvent is toluene or tetrahydrofuran or mixtures thereof.

Treatment of the reaction mixture and isolation of the desired product of formula I
a) extracting the reaction mixture with water while maintaining the pH in the range of 7.0 to 9.0, preferably in the range of 7.5 to 8.5 b) the pH in the range of 5.5 to 6.0 Extracting the aqueous phase with a water-immiscible organic solvent, preferably while maintaining a range of 5.5 to 5.7,
c) carried out applying a procedure comprising isolating the desired product of formula I from the organic phase and optionally d) crystallizing the desired product of formula I in an organic solvent be able to.

  The pH adjustment in step a) of the treatment procedure can take place using a common buffered aqueous solution, such as an aqueous sodium bicarbonate solution, whereas the pH in step b) can be achieved using an aqueous mineral acid solution, for example using an aqueous sulfuric acid solution. Can be adjusted.

  The water immiscible organic solvent is preferably toluene.

  Isolation in step c) usually occurs by partial evaporation of the organic solvent, after which the desired product is further purified by crystallization in a suitable organic solvent such as a mixture of toluene, isopropanol and heptane. Can do.

  The following examples illustrate the invention without limiting it.

Synthesis of Example (S, S) -3- [3-tert-butoxy-2- (9H-fluoren-9-yl-methoxycarbonylamino) -propionyl] -2,2-dimethyl-oxazolidine-4-carboxylic acid

Example 1:
A solution of 16.1 g N-hydroxysuccinimide and 40.0 g Fmoc-L-Ser (tBu) -OH in 200 mL THF was added to 26.0 g 1- (3- To the suspension of (dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added at 20 ° C. within 30-60 minutes. The resulting mixture was stirred at ambient temperature for 4 hours, then it was added to 8.75 g lithium hydroxide monohydrate, 6.1 g lithium carbonate and 33.2 g L-serine in 240 g water. Was added to the precooled (−5 ° C.) suspension within 30-45 minutes. The resulting mixture was allowed to warm to room temperature within 30 minutes and then stirred at this temperature for another hour. The mixture was then cooled to −5 ° C. and the pH was adjusted from 8.5 to 2.0-2.5 with about 150 g sulfuric acid (20% aqueous solution). The biphasic mixture was warmed to room temperature and then the lower aqueous layer was separated. The aqueous layer was extracted with 200 mL toluene. The combined organic layers were diluted with 150 mL toluene and then washed 5 times with 150 mL water. Water was removed from the organic layer by azeotropic distillation with toluene and THF. Anhydrous (<0.05%) toluene / THF solution (about 500 mL) was treated with 1.00 g of methanesulfonic acid. To the mixture was added a solution of 100 g 2,2-dimethoxypropane in 660 mL toluene within 6-10 hours. During the entire formulation, volatiles were removed by evaporation at a temperature of 20-28 ° C. under reduced pressure (80-30 mbar) while maintaining a constant reaction volume (about 600 mL). After complete addition, the mixture was concentrated to a final volume of about 500 mL and then treated with 1.35 g of triethylamine. Water (50 mL) was added and the layers were separated. The organic layer was treated with 250 g sodium bicarbonate (5% aqueous solution). The biphasic mixture (pH about 7.5) was warmed to 35-40 ° C. and stirred at this temperature for 30-45 minutes. The layers were separated and the organic layer was extracted 3 times with 70 g sodium bicarbonate (5% aqueous solution). The combined aqueous layer containing the product was treated with 360 mL of toluene at 35-40 ° C. and the pH was adjusted to 5.5 by dropwise addition of about 50 g of sulfuric acid (20% aqueous solution). The aqueous layer was separated and the organic layer was washed twice with 50 g of water. The resulting organic layer was cooled to ambient temperature and diluted with 100 mL water. The pH was adjusted to 4 by the addition of a few drops of sulfuric acid (20% aqueous solution). The lower aqueous layer was removed and the organic layer was washed twice with 80 g of water. The organic layer was concentrated to dryness. The residue was treated with 400 mL isopropanol and the resulting solution was concentrated to dryness. The residue was diluted with 90 mL isopropanol and 90 mL heptane and the mixture was warmed to 50 ° C. to achieve a clear solution. 400 mL of heptane was added within 3-4 hours. The mixture was then cooled to −10 ° C. within 13-16 hours and the resulting suspension was stirred at this temperature for at least 4 hours. The crystals are filtered off, washed with 80 mL pre-cooled heptane, dried at 40-50 ° C./<30 mbar and 31.6 g (60%) of (S, S) -3- [3-tert as colorless crystals. -Butoxy-2- (9H-fluoren-9-yl-methoxycarbonylamino) -propionyl] -2,2-dimethyl-oxazolidine-4-carboxylic acid was obtained (99.0% (m / m) by HPLC assay). ).

Example 2:
A solution of 16.1 g N-hydroxysuccinimide and 40.0 g Fmoc-L-Ser (tBu) -OH in 200 mL THF was added to 26.0 g 1- (3- To the suspension of (dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added at 20 ° C. within 30-60 minutes. The resulting mixture was stirred at ambient temperature for 4 hours, then it was added to 8.75 g lithium hydroxide monohydrate, 6.1 g lithium carbonate and 33.2 g L-serine in 270 g water. Added to pre-cooled (−5 ° C.) suspension within 30-60 minutes. The resulting mixture was allowed to warm to room temperature within 30 minutes and then stirred at this temperature for another hour. The mixture was then cooled to −5 ° C. and the pH was adjusted to 2.5 with 137 g sulfuric acid (20% aqueous solution). The biphasic mixture was warmed to room temperature and then the lower aqueous layer was separated. The aqueous layer was extracted with 210 mL toluene. The combined organic layers were diluted with 100 mL toluene and then washed 5 times with 130 mL water. Water was removed from the organic layer by azeotropic distillation with toluene and THF. The resulting toluene / THF solution (about 550 mL) was then treated with 1.00 g of methanesulfonic acid. Next, a solution of 134 g of 2,2-dimethoxypropane in 1040 mL of toluene was added to the mixture within 8-10 hours. During the entire formulation, volatiles were removed by evaporation at a temperature of 25-32 ° C. under reduced pressure (80-30 mbar) while maintaining a constant reaction volume (about 600 mL). After complete addition, the mixture was concentrated to a final volume of about 500 mL. The reaction mixture was treated with 250 g sodium bicarbonate (5% aqueous solution). The biphasic mixture was warmed to 35-40 ° C. and stirred at this temperature for 15 minutes. The layers were separated and the organic layer was extracted with 100 g sodium bicarbonate (5% aqueous solution). The combined aqueous layer containing the product was washed with toluene (150 mL). The aqueous layer was treated with 300 mL of toluene at 35-40 ° C. and the pH was adjusted to 5.7 by the dropwise addition of about 45 g of sulfuric acid (20% aqueous solution). The aqueous layer was then separated and the organic layer was washed 3 times with 80 g of water. The resulting organic layer containing the product was treated with 80 mL of water. The pH was adjusted to 4 by adding a few drops of sulfuric acid (20% aqueous solution). The lower aqueous layer was removed and the organic layer was washed twice with 80 g of water. The organic layer was concentrated to a residual volume of about 170 mL. The mixture was warmed to 55-60 ° C. and isopropanol (15 mL) was added. The resulting clear solution was treated with 300 mL heptane at 55-60 ° C. within 2-4 hours. The resulting suspension was cooled to 0 ° C. within 10 hours and stirred at this temperature for 3 hours. The crystals are filtered off, washed with 100 mL pre-cooled heptane, dried at 50 ° C./<30 mbar and 33.6 g (63%) of (S, S) -3- [3-tert-butoxy as colorless crystals. -2- (9H-fluoren-9-yl-methoxycarbonylamino) -propionyl] -2,2-dimethyl-oxazolidine-4-carboxylic acid was obtained (99.4% (m / m) by HPLC assay).

Claims (19)

formula

[Wherein R ¹ is a side chain of an α-amino acid, R ² is an amino protecting group, and R ³ and R ⁴ are independently selected from hydrogen or C _1-4 -alkyl (provided that Wherein R ³ and R ⁴ are not both hydrogen) , R ⁵ is hydrogen or methyl],
a) Formula

[Wherein R ¹ and R ² are as defined above] together with serine or threonine

(In which case water-soluble carbodiimide is used as an activator) and b) the formula

[Wherein R ³ and R ⁴ are independently selected from hydrogen or C _1-4 -alkyl, provided that both R ³ and R ⁴ are not hydrogen and R ^9a and R ^9b are independently C _{1-4 -} see including to implement the ring closure of the dipeptide of formula III in the presence of an acid catalyst with the compound represented by the alkyl is, furthermore,
a) extracting the reaction mixture with water while maintaining the pH in the range of 7.0 to 9.0;
b) extracting the aqueous phase with a water-immiscible organic solvent while maintaining the pH in the range of 5.5 to 6.0;
c) obtaining the desired product of formula I from the organic phase, and optionally
d) crystallizing the desired product of formula I in an organic solvent
A method comprising obtaining a target compound of formula I by a treatment procedure comprising : R ¹ is, valine, leucine, isoleucine, methionine, phenylalanine, asparagine, glutamine, glutamate, histidine, lysine, arginine, aspartic acid, alanine, serine, threonine, tyrosine, tryptophan, cysteine, is selected Ri by glycine and aminoisobutyric butyric acid The method according to claim 1, wherein the method is a side chain. ^3. A method according to claim 1 or 2, characterized in that R2 is selected from Fmoc, Z, Moz, Troc, Teoc and Voc. The method according to any one of claims 1 to 3 , wherein the water-soluble carbodiimide is EDC or a salt thereof.   The method according to claim 4, wherein EDC or a salt thereof is used together with HOSu. 6. The method according to any one of claims 1 to 5, characterized in that the ratio of serine or threonine: amino acid derivative of the formula II is selected from the range 1.5 to 4.0: 1. Conversion in step a), characterized in that it is carried out in the presence of an inorganic base, any one method according to claims 1-6.   8. Process according to claim 7, characterized in that the inorganic base is selected from alkali carbonates or alkali hydroxides and mixtures thereof. 9. Process according to any one of claims 1 to 8, characterized in that the conversion in step a) is carried out at a pH in the range of 7.5 to 9.5. The process according to any one of claims 1 to 9, characterized in that the transformation in step a) is performed at a temperature in the range of -10 ° C to 25 ° C. Process according to any one of claims 1 to 10, characterized in that the reaction mixture is acidified with mineral acid after the conversion in step a). 12. Process according to any one of claims 1 to 11, characterized in that the dipeptide of formula III is used directly in process step b) without being isolated. 13. Process according to any one of claims 1 to 12, characterized in that the compound of formula IV used for ring closure in step b) is 2,2-dimethoxypropane.   The process according to claim 13, characterized in that 2,2-dimethoxypropane is continuously added to the reaction mixture and the methanol generated is distilled off continuously in parallel. The acid catalyst for ring closure in step b) is selected from methanesulfonic acid, (+) camphor-10-sulfonic acid, p-toluenesulfonic acid, pyridinium p-toluenesulfonate. The method of any one of 1-14. The process according to any one of claims 1 to 15, characterized in that the ring closure in step b) is carried out in the presence of toluene or tetrahydrofuran or mixtures thereof. Step ring closure in b) is characterized in that it is carried out at a temperature in the range of 15 ° C. to 35 ° C., The method of any one of claims 1 to 16. Water-immiscible organic solvent, characterized in that toluene, the process of claim 1.   19. A process according to claim 18, characterized in that the target product is crystallized from a mixture of toluene, isopropanol and heptane.